Site-directed mutagenesis of human vasoactive intestinal peptide receptor subtypes VIP1 and VIP2: evidence for difference in the structure-function relationship.
Vasoactive intestinal peptide (VIP1 and VIP2) receptors belong to the new class II subfamily of G protein-coupled receptors. We investigated here human VIP1 and VIP2 receptors by mutating in their extracellular domains all amino acid residues that are conserved in VIP receptors but are different in other members of their subfamily. They are present in 1) the N-terminal domain, i.e., E36, I43, S64, D132 and F138 in the VIP1 receptor and E24, I31, S53, D116 and F122 in the VIP2 receptor; 2) the second extracellular loop, i.e., T288 and S292 in the VIP1 receptor and T274 and S278 in the VIP2 receptor. These residues were changed to alanine (A), and cDNAs were transfected into Cos cells. For the VIP1 receptor, no specific 125I-VIP binding could be detected in cells transfected with the E36A mutant, whereas other mutants exhibited Kd values similar to that of the wild-type receptor, with the exception of S64A, for which a 3-fold increase of Kd was observed. For the VIP2 receptor, no specific 125I-VIP binding could be observed with the E24A mutant, whereas other mutants exhibited dissociation constants similar to that of the wild-type receptor, with the exception of I31A and T274A mutants, for which a 11- and 5-fold increase of Kd was observed, respectively. cAMP production experiments provided evidence that the E36A VIP1 receptor and the E24A VIP2 receptor mutants mediated almost no response upon VIP exposure. For the I31A and T274A mutants of the VIP2 receptor and the S64A mutant of the VIP1 receptor, the EC50 values of VIP for stimulating cAMP production were increased 35, 8 and 3 times as compared with that observed for the wild-type receptor, respectively. Immunofluorescence studies indicated that all mutants were normally expressed by Cos cells. These data provide the first evidence for differences in the structure-function relationship of VIP1 and VIP2 receptors.
Nicole
P
P
Unité de Neuroendocrinologie et Biologie Cellulaire Digestives, Institut National de la Santé et de la Recherche Médicale, INSERM U410, Faculté de Médecine Xavier Bichat, 75018 Paris, France.
Du
K
K
Couvineau
A
A
Laburthe
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000378:metabolism; D005110:Extracellular Space; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011869:Radioligand Assay; D018005:Receptors, Vasoactive Intestinal Peptide / Q000737:chemistry* / Q000378:metabolism; D051239:Receptors, Vasoactive Intestinal Peptide, Type II; D051238:Receptors, Vasoactive Intestinal Polypeptide, Type I; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D014162:Transfection
D018005:Receptors, Vasoactive Intestinal Peptide; D051239:Receptors, Vasoactive Intestinal Peptide, Type II; D051238:Receptors, Vasoactive Intestinal Polypeptide, Type I; C495509:VIPR2 protein, human; D000242:Cyclic AMP